Antibody-drug conjugates

ABSTRACT

Immunoconjugates of antibodies or antigen-recognizing fragments of antibodies and monovalent cytotoxic drug derivatives make use of β-alanine derived linkers wherein the antibody or fragment thereof is attached to the linker&#39;s carboxy group via an ester or amide group and the drug is attached through the linker&#39;s 2-position methylene group. Intermediates, compositions and methods of use also are provided.

FIELD OF THE INVENTION

The present invention belongs to the fields of organic chemistry,pharmaceutical chemistry, and immunology, and provides immunoconjugateswhich are vehicles for targeting large doses of pharmaceutically-activecompounds to undesirable cells, tissues, and mammalian pathogens.

Immunoconjugates of the present invention consist of antibodies,preferably monoclonal antibodies which act as targeting agents, apharmaceutically-active compound which has activity against the cell,tissue, or host in need of treatment, and an organic compound whichjoins or links ("linker") such antibodies with suchpharmaceutically-active compounds. Intermediates for the preparation ofthe immunoconjugates are also provided.

BACKGROUND OF THE INVENTION

In recent years, pharmaceutical chemists have worked to provide morespecific and potent drugs for the treatment of disease. In the case ofcancer and other disease which function by the creation of specificabnormalities of cells, most of the useful drugs have been of thecytotoxic type which function by killing the abnormal cell. Such drugsare quite potent and may be hazardous to the recipient, evenlife-threatening.

In an attempt to direct a drug to particular cells, tissues, or pathogenwithin a host, efforts have been made to develop a mechanism fortargeting such drugs, particularly highly cytotoxic drugs, directly tothe afflicted area or pathogen, without administering a whole-bodydosage. However, no antibody-drug conjugate has been approved fortherapeutic use. The present invention expands the scope ofimmunoconjugate technology by providing novel immunoconjugates usefulfor cell-/tissue-/pathogen-specific drug delivery in mammals andintermediates thereto.

SUMMARY OF THE INVENTION

The present invention provides a physiologically-acceptable drugconjugate of formula I ##STR1## wherein R¹ is C₁ -C₄ alkyl;

R² is a monovalent drug derivative having a reactively-available amino,hydroxy or thiol function;

m is an integer from 1 to 10; and

Ab is an antibody, or antigen-recognizing fragment thereof, whichrecognizes an antigen associated with a cell, tissue, or pathogen towhich delivery of the drug is desirable.

The invention also provides intermediates of the following formulae##STR2## wherein R¹ is C₁ -C₄ alkyl;

R⁴ is a carboxy protecting group;

R⁵ is H₂, ═C(OH)₂, ═CHOR⁶, or ═CHSR⁶ ; and

R⁶ is C₁ -C₄ alkyl; and ##STR3## wherein R¹ is C₁ -C₄ alkyl;

R² is a monovalent drug derivative having a reactively-available amino,hydroxy or thiol function; and

R⁷ is H, a carboxy protecting group, or a carboxy activating group, or amoiety which completes a salt of the carboxy group.

The present invention further provides pharmaceutical compositionscomprising an immunoconjugate of the invention and aparenterally-administrable medium. Also provided are treatment methodscomprising the parenteral administration of an immunoconjugate of theinvention to a patient in need of treatment with a pharmaceuticallyactive compound (drug).

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to novel immunoconjugates, intermediatesthereto, pharmaceutical compositions and methods of use.

Throughout the present document, all temperatures are in degreescelsius. All expressions of percentage, concentration and the like arein weight units, unless otherwise stated. All references toconcentrations and dosages of drug conjugates are in terms of the amountor concentration of the drug contained in the conjugate.

In the above formulae, the general and specific chemical terms used havetheir normal meanings in organic and, especially, amino acid chemistry.

For example, the term "C₁ -C₄ alkyl" refers to straight or branchedaliphatic chains of 1-4 carbon atoms including methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

The carboxy protecting groups of R⁴ and R⁷, when R⁷ is neither H nor anactivating group, denote groups which generally are not found in finaltherapeutic conjugates, but which are intentionally introduced during aportion of the synthetic process to protect a group which otherwisemight react in the course of chemical manipulations, and is then removedat a later stage of the synthesis. Since compounds bearing suchprotecting groups are of importance primarily as chemical intermediates,their precise structure is not critical. Numerous reactions aredescribed in a number of standard works including for example,Protective Groups in Organic Chemistry, Plenum Press, (London and NewYork, 1973); Green, T. W., et al. (eds), Protective Groups in OrganicSynthesis, Wiley (New York, 1981); and Schroeder, et al. (eds.), ThePeptides,Vol. I, Academic Press (London and New York, 1965 ).

Preferred R⁴ and R⁷ carboxy protecting groups include C₁ -C₄ alkyl,phenyl which maybe substituted, and benzyl which may be substituted. Theterm "phenyl which may be substituted" denotes an unsubstituted orsubstituted phenyl residue, optionally having one or two substituentsselected from halo (bromo, chloro, fluoro, and iodo), nitro, C₁ -C₄alkyl and C₁ -C₄ alkoxy. The term "benzyl which may be substituted"denotes an unsubstituted or substituted benzyl ring, optionally havingone or two substituents selected from halo, nitro, C₁ -C₄ alkyl and C₁-C₄ alkoxy. The term "C₁ -C₄ alkoxy" represents a C₁ -C₄ alkyl groupattached through an oxygen bridge such as, for example, methoxy, ethoxy,n-propoxy, isopropoxy, and the like. Unsubstituted and substitutedbenzyl are especially preferred carboxy protecting groups.

The term "carboxy activating group" refers to groups used in syntheticorganic chemistry which increase the reactivity of a carboxy group. Suchgroups are well known in the organic synthetic art and, when bound withthe single-bonded oxygen of a carboxy group, include groups such as, forexample, benzenesulfonyloxy, methanesulfonyloxy, toluenesulfonyloxy,phthalimidyloxy, succinimidyloxy, chloro, benzotriazolyloxy, bromo,azido, and the like. Preferred carboxy activation groups includebenzotriazolyloxy and phthalimidyloxy while succinimidyloxy isespecially preferred.

The immunoconjugates of the present invention are composed ofantibodies, drugs of certain chemical classes, and organic chemicalgroups which link the antibodies and drugs. The invention also providesβ-alanine derivative intermediates which are useful for the preparationof such immunoconjugates.

The Antibody

It will be understood that the function of the present drug conjugatesis determined by the biological efficacy of the drug and the antigenicselectivity of the antibody. An antibody is chosen which will recognizean antigen associated with a cell, tissue or pathogen to which theparticular drug is delivered to the benefit of a patient. For example,if the drug is an antineoplastic, then an antibody which recognizes anantigen associated with tumor cells would be chosen.

In addition to antineoplastic agents, other antiproliferative agentssuch as those used in the treatment of cardiovascular disease may beselected to be "linked" with site-specific antibodies. For example, itis well established that restinosis frequently follows angioplasty. Thesite-specific delivery of antiproliferative drugs to areas whereatheriosclerotic plaque was removed would be useful in retarding orpreventing reocclusion.

Likewise, an antibacterial or antiviral drug would be linked to anantibody which would recognize a respective bacterium or virus.

Depending upon the characteristics of the drug to be used, it may bepreferred in a given case to choose an antibody which is internalized bythe cell, or it may be preferred to use an antibody or antigen-bindingfragment thereof which remains on the cell surface by recognizing asurface antigen.

The source of the antibody is not critical to the present invention. Itmay be chosen from any class or subclass of immunoglobulin includingIgG, IgA, IgM, IgE and IgD. Similarly, the species of origin is notcritical so long as the antibody targets a cell where the effect of thedrug is useful.

In the present state of the art, monoclonal antibodies and theirfragments are frequently used in drug conjugates, and use of them ispreferred in the present invention. However, polyclonal antibodies andtheir fragments are not excluded.

Newer types of antigen binding molecules can be produced by recombinanttechnology. See, e.g., Hodgson, Bio/Technology, 4:421-25 (1991). Morespecifically, genetically engineered antibodies which retain the epitopespecificity of monoclonal antibodies are now known in the art andprovide a less immunogenic molecule. Such genetically engineeredantibodies are also embraced by the present invention.

Furthermore, chimeric antibodies are described in U.S. Pat. No.4,816,567 (Cabilly), which is herein incorporated by reference.

A further approach to production of genetically engineered antibodies isprovided in U.S. Pat. No. 4,816,397 (Boss) which is also hereinincorporated by reference. The approach of U.S. Pat. No. 4,816,397 hasbeen further refined as taught in European Patent Publication 0 239 400which published September 30, 1987. The teachings of European PatentPublication 0 239 400 (Winter) are the preferred format for the geneticengineering of monoclonal antibodies which are used as components of theimmunoconjugates of the invention. The Winter technology involves thereplacement of complementary determining regions (CDRs) of a humanantibody with the CDRs of a murine monoclonal antibody, therebyconverting the specificity of the human antibody to the specificity ofthe murine antibody which was the source of the CDR regions. The CDRtechnology provides a molecule which contains minimal murine sequencesand, thus, is less immunogenic.

Single chain antibody technology is yet another variety of geneticallyengineered antibody which is well known in the art. See, Bird, R. E., etal., Science, 243.: 423-426 (1988). The single chain antibody technologyinvolves joining the binding regions of heavy and light chains with apolypeptide sequence to generate a single polypeptide having the bindingspecificity of the antibody from which it was derived.

The aforementioned genetic engineering approaches provide the skilledartisan with numerous means to generate molecules which retain thebinding characteristics of the parental antibody while affording a lessimmunogenic format. Thus, genetically engineered antibodies may beobtained and used in the present invention.

The origin and nature of the antibody is not otherwise critical, so longas it targets the cell, tissue or pathogen to be treated and is not, initself, toxic to the patient. Some discussion of the method ofevaluating antibodies and conjugates will be provided for convenience.

First, the antibody should be produced by a hybridoma or modifiedmicroorganism which is sufficiently stable to allow preparation ofreasonable quantities of antibody. The antibody itself should beamenable to purification and, in particular, should be sufficientlywater-soluble to allow chemical manipulations at reasonableconcentrations.

Next, conjugates prepared with the candidate antibody are evaluated forantigen-binding capacity. Skilled artisans will appreciate the ease withwhich any diminution in antigen binding activity can be determined.Competitive radioimmunoassays (RIAs) and flow cytometry are two of themost convenient means for determining whether a conjugate has reducedbinding capacity relative to the pristine antibody. A modest reductionfrom the binding capacity of the free antibody is expected andacceptable. Then, the conjugate is tested to determine its in vitropotency, such as cytotoxicity in the case of anticancer drugs, againstantigen-positive cells. An effective conjugate can have potency somewhatless than the free drug in the same assay because of its ability tobring a high concentration of drug to the cell. A conjugate which isaccepted in the first two tests is then evaluated in a nude mouse humantumor xenograft model as taught by Johnson and Laguzza, Cancer Res., 47:3118-22 (1987). The candidate conjugate should be tested, for example,in nude mice against the free drug, a mixture of free drug and freeantibody, and a conjugate with a non-targeting immunoglobulin. Theconjugate should generally exhibit improved potency or safety. Doseranging studies should be carried out in the xenograft model.

Conjugates which are potent in the xenograft model are submitted totests in animals which are known to express the antigen of interest in apattern similar to that seen in humans. If the conjugate produces asignificant degree of binding to the antigen in such tests, and if it isreasonably free of toxicity at doses predicted by the xenograft model tobe therapeutic, the candidate conjugate can be considered to havetherapeutic potential.

It will be understood that properly chosen fragments of antibodies havethe same effect as the intact antibody. Skilled artisans will recognizethe utility which the proteolytic enzymes papain and pepsin possess forcleaving immunoglobins into fragments which are bivalent or monovalent,respectively. Thus, in the practice of this invention, fragments ofantibodies, particularly F(ab')2 fragments, which recognize an antigenassociated with the cell to be treated, may be just as useful as areintact antibodies. Fab fragments are also useful.

The mechanism by which the linker group reacts and attaches to theantibody depends on the R⁷ group in the linker of formula III wherein R⁷is a carboxy activating group. The linking mechanism of the R⁷ groupwill be explained below in detail in the section on synthesis of theconjugates.

Formula I indicates that from 1 to 10 linker-drug moieties (derivatizeddrug) are attached to each molecule of antibody. Of course, the numberof such moieties per antibody molecule is an average number because agiven batch of conjugate will necessarily contain molecules having arange of ratios of derivatized drug to antibody. The most efficient useof the expensive antibody is obtained, of course, when a number ofmolecules of drug are attached to each antibody molecule. However, theattachment of an excessive number of molecules of derivatized drugusually has an adverse effect on the antibody's ability to recognize andbind to its antigen, so a compromise value for m must be found.

Generally, the preferred average value for m is from 3 to 6. Conjugationratios are easily determined by measuring the absorbance of theimmunoconjugate at wave-lengths selected to detect the drug, the linker,peptide bonds, et cetera, and then deduce from the absorbance data andthe extinction coefficients for the various components the averageamount of drug per antibody.

A great number of antibodies are available to immunologists for use inthe present invention, and further useful antibodies are being disclosedin every issue of the relevant journals. It is impossible, and entirelyunnecessary, to give an exhaustive listing of antibodies which can beapplied in the practice of this invention. Immunologists and chemists ofordinary skill are entirely able to choose antibodies from sources suchas the catalogue of the American Type Culture Collection (ATCC),Rockville, Md., U.S.A., and Linscott's Directory of Immunological andBiological Reagents, published by Linscott's Directory, 40 Glen Drive,Mill Valley, Calif., U.S.A., 94941. Thus, it is a simple matter for theartisan in the field to choose an antibody against virtually anydeterminant, such as tumor, bacterial, fungal, viral, parasitic,mycoplasmal, or histocompatibility antigens, as well as pathogen surfaceantigens, toxins, enzymes, allergens and other types of antigens relatedto physiologically important cells and tissues.

The most preferred use of the present invention is in the delivery ofcytotoxic drugs to cancer cells, particularly including squamouscarcinoma cells, adenocarcinoma cells, small cell carcinoma cells,glyoma cells, melanoma cells, renal cell carcinoma cells, transitionalcell carcinoma cells, sarcoma cells, cells of supporting tumorvasculature, and cells of lymphold tumors such as leukemias andlymphomas. Appropriate antibodies for the targeting of all such cellsare available, and sources can be located in Linscott. Alternatively,the necessary hybridomas for the production of such antibodies byconventional methods are obtainable through the ATCC and other cell linecollections.

A number of presently known antibodies are particularly interesting foruse in the anticancer aspect of the present invention. Preferredspecific antibodies, for example, are VX007B, CC83, CC49 and D612.

Another interesting antibody is KS1/4, first disclosed by Varki, et al.,Cancer Research, 44:681-86 (1984). A number of plasmids which comprisethe coding sequences of the different regions of monoclonal antibodyKS1/4 are now on deposit and can be obtained from the ATCC, Peoria,Ill., U.S.A. The plasmids can be used by those of ordinary skill toproduce chimeric antibodies by recombinant means, which antibodies bindto a cell surface antigen found in high density on adenocarcinoma cells.The construction of such antibodies is discussed in detail in U.S. Pat.No. 4,975,369. The following plasmids relate to KS1/4.

Plasmid pGKC2310, the coding sequence of the light chain, the signalpeptide associated with the light chain, and the 5' and 3' untranslatedregions; isolated from E. coli K12 MM294/pGKC2310, NRRL B-18356.

Plasmid pG2A52, the coding sequence of the heavy chain, the codingsequence of the signal peptide associated with the heavy chain, and the5' and 3' untranslated regions; isolated from E. coli K12 MM294/pG2A52,NRRL B-18357.

Plasmid CHKC2-6, the coding sequence of the light chain variable region,the coding sequence of the signal peptide associated with the lightchain, and a sequence encoding the light chain constant region of humanIgG; isolated from E. coli K12 DH5/CHKC2-6, NRRL B-18358.

Plasmid CHKC2-18, the coding sequence of a derivative light chainvariable region, the coding sequence of the signal peptide associatedwith the light chain, and a sequence encoding the light chain constantregion of a human IgG; isolated from E. coli K12 DH5/CHKC2-18, NRRLB-18359.

Plasmid CH2A5, the coding sequence of the heavy chain variable region,the coding sequence of the signal peptide associated with the heavychain, and a sequence encoding the heavy chain constant region of humanIgG1; isolated from E. coli K12 MM294/CH2A5, NRRL B-18360.

Plasmid CH2A5IG2, the coding sequence of the heavy chain variableregion, the coding sequence of the signal peptide associated with theheavy chain, and a sequence which encodes the heavy chain constantregion of human IgG2; isolated from E. coli K12 DH5/CH2A5IG2, NRRLB-18361.

Plasmid CH2A5IG3, the coding sequence of the heavy chain variableregion, the coding sequence of the signal peptide associated with theheavy chain, and a sequence encoding the heavy chain constant region ofhuman IgG3; isolated from E. coli K12 DH5/CH2A5IG3, NRRL B-18362.

Plasmid CH2A5IG4, the coding sequence of the heavy chain variableregion, the coding sequence of the signal peptide associated with theheavy chain, and a sequence encoding the heavy chain constant region ofhuman IgG4; isolated from E. coli K12 DH5/CH2AIG4, NRRL B-18363.

Antibody 5E9C11, produced by an ATCC hybridoma, HB21, recognizes thetransferrin receptor which is expressed by many tumors. An antibodynamed B72.3, available from the National Cancer Institute, recognizesantigens expressed by both breast and colon carcinoma.

Two interesting antibodies with reactivities against non-tumor antigensare OKT3 and OKT4. These antigens bind to peripheral T-cells and humanT-helper cells, respectively.

Additional sources of antibodies useful for various therapeutic purposesinclude, for example, the following. Antihuman lymphocyte and monocyteantibodies, useful for immune modulation and tumor therapy, are producedby ATCC cultures, HB2, HB44, HB78 and HB136. An antitransferrin receptorantibody, useful for tumor therapy, is produced by ATCC culture HB84.ATCC culture HB8059 produces an antibody against colorectal carcinomamonosialoganglioside, and culture B8136 produces an antibody againstmature human T-cell surface antigen, useful for immune modulation andT-cell leukemia therapy.

Furthermore, ATCC hybridoma HB9620 will produce a convenientanticarcinoembryonic antigen called CEM231.6.7.

Schlom, et al. have disclosed a number of interesting antibodies whichhave affinities for tumor-related antigens. In particular, the followingarticles and patents by that group are important.

Cancer Research, 50: 1291-98 (1990)

Cancer Research, 45: 5769-80 (1985)

International J. Cancer, 49: 598-607 (1989)

U.S. Pat. No. 4,522,918

U.S. Patent No. 4,612,282

European Patent Publication 0,394,277

European Patent Publication 0,225,709

The antibodies taught by the Schlom group which are functional in thecontext of the present invention include those with the designationsD612, COL- 1 through COL- 15, CC-1, CC-8, CC-9, CC-11, CC-14, CC-15,CC-20, CC-26, CC-29, CC-30, CC-41, CC-46, CC-48, CC-49, CC-52, CC-55,CC-57, CC-60, CC-63, CC-66, CC-72, CC-74, CC-78, CC-83, CC-87, CC-90,CC-92.

An immunologist or one knowledgeable in the art of drug targeting, withthe assistance of the commonly known publications in the field and theabove guiding examples and description, can readily choose an antibodyfor the targeting of any appropriate drug to any desired cell to betreated with that drug.

Methods of producing and purifying monoclonal antibodies are also wellknown to one skilled in the art of immunology. For a review of methodsfor culturing hybridomas, generating ascites, and purifying antibodies,see, Mishell, B., et al. (eds), Select Methods in Cellular Immunology,W. H. Freeman and Company (New York, 1980).

The Drug

It will be understood that the essence of the present invention is themethod of linking a monovalent drug derivative (hereinafter drug) andantibody by means of the above-described β-alanine derived linkers, andthat neither the drug nor the antibody is a limitation of the presentinvention. The linkers of the present invention, accordingly, may be andare beneficially used when applied to drugs of any therapeutic orprophylactic purpose, limited only by the necessity for the drug topossess a chemical function with which the β-alanine derivative canlink, and the necessity for the antibody to target a cell where the drugis beneficial. The methylidene linking mechanism provided by the presentinvention requires the drug to have a reactively-available amino,hydroxy or thiol function. Furthermore, the drug must be of such anature that the reaction of the reactively-available function with thelinker does not destroy the activity of the drug.

Accordingly, the present linker invention may be used in connection withdrugs of substantially all classes including, for example,antibacterials, antivirals, antifungals, anticancer agents,antimycoplasmals, and the like. The drug conjugates so constructed areeffective for the purpose for which the corresponding drugs areeffective, and have superior efficacy because of the ability, inherentin the antibody, to transport the drug to the cell, tissue or pathogenwhich would respond to such drug therapy.

Drugs and other compounds which may be subjected to drug conjugation aredisclosed in U.S. Pat. Nos. 5,010,176 and 4,671,958, and the disclosureconcerning drugs of these patents is herein incorporated by reference.

As previously stated, the drug is reacted with the linker through areactively-available amino, hydroxy or thiol function on the drug. Thereactively-available functionality of the drug may originally be part ofthe drug or may be introduced for the purpose of forming a derivatizeddrug. The term "reactively-available amino function" includes aminogroups which are part of hydrazides, hydrazines, carbamates, and thelike, as well as amino groups simply attached to a carbon-hydrogenstructure. An amino group may have a third small substituent on itproviding the group does not create steric hindrance which preventsreaction with the β-alanine derivative structure. Such groups may be,for example, straight- or branched-chain alkyl groups and the like.

Similarly, the terms "reactively-available hydroxy function" and"reactively-available thiol function" include simple alcohols andcarboxylic acid, and thioic acid, respectively.

While the use of drugs of any chemical type having areactively-available function and any therapeutic or prophylacticefficacy is included in the present invention, it is preferred to usedrugs which have an amino function available for reaction. It is morepreferred to use drugs wherein the amino group is part of a hydrazine orhydrazide moiety.

The most preferred efficacy class of drugs for use in the presentinvention is the class of cytotoxic drugs and, particularly, those whichare used for cancer therapy. Such drugs include, in general, alkylatingagents, antiproliferative agents, tubulin binding agents, and the like.

Preferred classes of cytotoxic agents include, for example, thedaunomycin family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the pteridine family of drugs,and the podophyllotoxins.

Particularly useful members of those classes include doxorubicin,daunorubicin, aminopterin, methotrexate, lometrexol, methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine,or derivatives thereof, and the like. Of these, vinblastine andderivatives thereof, particularly desacetylvinblastine and derivativesthereof, and doxorubicin and derivatives thereof are especiallypreferred drugs. Desacetylvinblastine hydrazide and doxorubicin arehereinafter characterized as DAVLB-NHNH₂ and DOX-NH₂ ; respectively.

It will be understood that chemical modifications may be made by theordinarily skilled artisan to the preferred and generally describedcompounds in order to make reactions of them more convenient.

It will also be understood that preferred immunoconjugates are preparedfrom the preferred drugs.

The Intermediates

The intermediate β-alanine derivatives of the present invention(formulas II and III) are the intermediates which are reacted with theantibody and the drug and, thus, are the precursors of the linker whichjoins the antibody and the drug. Accordingly, the preferred β-alaninederivative intermediates confer their structure on the preferredimmunoconjugates of the present invention.

The intermediates are derived of β-alanine and are prepared according toprocesses known or readily imagined by ordinarily skilled organicchemists.

Synthesis Of the Intermediate Beta-alanine Derivatives

The intermediate β-alanine derivatives of the present invention areprepared by processes known to one skilled in the organic chemical artusing readily available reagents.

In the first step to prepare the novel intermediates of the presentinvention, a protected β-alanine compound of formula IV is reacted witha diketene of formula V in an inert solvent or mixture of solvents inthe presence of a base such as N-methylmorpholine and the like. One willrecognize that the length of the R⁸ substituent of formula V (C₁ -C₃alkyl) dictates the length of the R¹ substituent in compounds of formulaI, II, and III. This reaction, which produces compounds of formula IIa,is known in the art and is depicted below in Equation 1.

Equation 1 ##STR4## wherein R¹ is C₁ -C₄ alkyl;

R^(4a) is a carboxyl protecting group;

R^(5a) is H₂ ; and

R⁸ is H or C₁ -C₃ alkyl.

Suitable solvents are any polar solvent, or mixture of solvents, whichwill remain inert or substantially inert under reaction conditions.Preferably, a mixture of solvents containing methylene chloride anddimethylformamide DMF) is used in this reaction. A preferred ratio ofthese preferred solvents is 5:1, respectively.

The amount of reactants and reagents, the temperature employed, and thelength of time that is required to effect this reaction is known in theart and is apparent to a skilled organic chemist (see, e.g., Example 1).

The second step in synthesizing the compounds of the present inventionpreferably requires reacting a compound of formula IIa with anappropriate trialkylorthoformate such as triethylorthoformate and thelike, in the presence of a Lewis acid such as zinc chloride and thelike, and an alcohol scavenger such as acetic anhydride and the like, toform an alkoxymethylidene derivative of formula IIb. This reaction iscarried out at an elevated temperature, in the range of 100°-200°, andis completed in a few hours time. Equation 2 below depicts thispreferred reaction. Alternatively, R⁵ may be alkylthiomethylidene byusing procedures well known in the art.

Equation 2 ##STR5## wherein R¹, R^(4a) and R^(5a) are as defined above;##STR6## wherein R¹ and R^(4a) are as defined above; and

R^(5b) is ═C(OH)₂, ═CHOR⁶, or ═CHSR⁶ ; and

R⁶ is C₁ -C₄ alkyl.

In each reaction of the present invention, no unusual excess amount ofstarting compounds is necessary. As is ordinarily the case in organicchemistry, it is advisable to use a moderate excess of comparativelyinexpensive reactants in order to assure that more expensive reactantsare fully consumed. This rule is particularly true in the case of thereactions with antibodies which typically are expensive and difficult toprepare and purify. In general, however, amounts of excess reactants maybe utilized for the purpose of maximizing the economy of the process,bearing in mind the cost of the ingredients as well as throughput of theequipment. Thus, it is unnecessary to use excess amounts of reactantsmerely to force the reactions to occur.

In the third step of the process used to prepare intermediates of thepresent invention, formula IIb compounds are catalytically hydrogenated,in the presence of a suitable solvent, to form compounds of formula IIc.In this well known reaction, the purpose of which is to remove theR^(4a) carboxyl protecting group and form the acid thereof,1,4-cyclohexadiene is the preferred reducing agent.

Suitable hydrogenation catalysts include noble metals and oxides such aspalladium, platinum and rhodium oxide on a support such as carbon orcalcium oxide. However, palladium-on-carbon is preferred.

Solvents for this reaction are those solvents or mixture of solventswhich remain inert throughout the reaction. Typically, alcohols such asmethanol, 1-propanol, 2-propanol and, especially, ethanol, are suitablesolvents.

In addition to the free acid, the R^(4a) carboxylic acid may be furtherderivatized to its salt form. The salts are formed with any moietycapable of forming a physiologically-acceptable salt of the carboxylicacid. Alkali metal and hydrohalide salts are particularly appropriate.Thus, the sodium, potassium and lithium salts, as well as thehydrochloride, hydrobromide and hydrofluoride salts, are particularlyuseful in the practice of the present invention. Other salts acceptablein pharmaceutical chemistry, however, are also useful. For example,amine salts such as triethylamine, triethanolamine, ethyldimethylamineand the like are useful, as are quaternary ammonium salts includingtetraalkylammonium salts, (benzyl or phenyl) trialkylammonium salts andthe like. Among ammonium salts, tetrabutylammonium,benzyltrimethylammonium, and tetramethylammonium are typical andpreferred salts. Pharmaceutical chemists frequently use salts ofcarboxylic acids and the present salts, wherein R^(4b) is a salt-formingmoiety, may be prepared with any base which forms aphysiologically-acceptable salt.

This aspect of the process is depicted in Equation 3.

Equation 3 ##STR7## R¹ and R⁶ are as defined above; R^(5b) is ═C(OH)₂,═CHOR⁶, or ═CHSR⁶ ; and

R⁴ is H or a moiety which completes a salt of the carboxylic acid.

Especially preferred substituents for formula II compounds is asfollows:

    ______________________________________            SUBSTITUENT    COMPOUND  R.sup.1   R.sup.4 R.sup.5    ______________________________________    IIA       methyl    benzyl  --H.sub.2    IIb       methyl    benzyl  ethyloxymethylidene    IIc       methyl    H       ethyloxymethylidene    ______________________________________

Compounds of formulae IIa, IIb, and IIc are each novel, are useful forthe preparation of immunoconjugates of the present invention, and arecollectively incorporated into compounds of formula II herein.

Reactions with Intermediates and Drums

The intermediates of formula IIc are reacted with drugs under conditionswhich will allow the alkylthiol, alcohol or alkoxy group of the R^(5b)substituent to be cleaved, and the remaining methylidenyl group to reactwith the reactively-available amino, hydroxy or thiol function of thedrug. In general, the reactions are carried out at temperatures fromabout -30° to about 50°, in inert organic solvents or in aqueousmixtures of such organic solvents, and usually in the presence of mildbases such as alkali metal bicarbonates, carbonates and hydroxides.Generally, the reactions are quantitative and require no unusual excessamounts of reactants. This reaction provides the derivatized drugcompounds of formula IIIa ##STR8## wherein R¹ is C₁ -C₄ alkyl;

R² is a monovalent drug derivative having a reactively-available amino,hydroxy or thiol function; and

R^(7a) is H or a moiety which completes a salt of the carboxylic acidor, in the alternative, a carboxy protecting group which will later bereduced to form the acid or a moiety which completes a salt of thecarboxylic acid.

Isolation of the product may require chromatography under high pressureor other sophisticated procedures because it usually is important topurify the derivatized drug with considerable care. Because thederivatized drug is later reacted with the antibody to completepreparation of the immunoconjugate, any reactive impurity whichaccompanies the derivatized drug may consume reactive sites on theantibody.

The removal of the carboxy protecting group from a formula IIb compound,in Equation 3, may be accomplished either before or after the β-alaninederivative intermediate is reacted with the drug. If it is necessary touse protecting groups on the drug, it may well be possible for thosegroups to be removed under the same conditions which remove theabove-mentioned protecting group. Thus, the drug may first be reactedwith a compound of formula IIb and both the drug and the β-alaninederived intermediate are deprotected, or a formula IIb compound is firstdeprotected and a formula IIc compound is reacted with the drug, forminga compound of formula IIIa.

Carboxy Activating Groups

In preparation for reacting the derivatized drug with the desiredantibody, the carboxylic acid or salt moiety of formula IIIa is firstactivated. The process of activating a carboxylic acid (where R^(7a) offormula IIIa compounds is H or a salt moiety thereof) is well known inthe art and is generally accomplished by use of conventionalesterification reagents such as carbodiimides, particularlydicyclohexylcarbodiimide, and an activating group such asN-hydroxysuccinimide and the like. Compounds of formula IIIb result fromthis reaction ##STR9## wherein R¹ and R² are as described above; and

R^(7b) is a carboxy activating group.

Especially preferred R¹ and R⁷ groups for formula IIIa compounds aremethyl and hydrogen, respectively; especially preferred R¹ and R^(7b)substituents for formula IIIb compounds are methyl andN-hydroxysuccinimide, respectively.

Reactions with activating groups are carried out in an inert organicsolvent such as, for example, dioxane, tetrahydrofuran, chlorinatedhydrocarbons, and the like or a mixture thereof. These reactionsgenerally may be performed at moderate temperatures in the range fromabout 0° to about 50°.

Compounds of formula IIIa and IIIb are each novel, are useful for thepreparation of immunoconjugates of the present invention, and arecollectively incorporated into compounds of formula III herein.

Synthesis of the Immunoconjugates

Once a derivatized drug is made, as is described above (formula III), itis reacted with the antibody as the final step in preparing theconjugate.

The primary concern in choosing the conditions under which to react thederivatized drug with the antibody is maintaining the stability of theantibody. The reaction must be carried out in an aqueous medium of acomposition which will not harm the antibody. A particularly suitableaqueous medium is a sodium carbonate buffer solution, in which theconcentration of carbonate ion is in the range of about 0.05 to about0.5 molar. The reaction also may be carried out in slightly acidicphosphate buffers, and physiological phosphate buffered saline (ppbs)and the like. Although the reaction medium should be aqueous, smallamounts of organic solvents in the reaction medium are not harmfulproviding the solvents do not have a tendency to damage the antibody.

The pH of the reaction medium should be maintained at a range from about7 to about 9, and reaction of the derivatized drug with the antibodymust be carried out at temperatures from about 4° to about 40°.

Because the solubility of antibodies is not great, the concentration ofthe antibodies in the reaction medium should be maintained at relativelylow concentrations. For example, the concentration of antibody isusually in the range of from about 5 to about 25 mg per mL of aqueousmedium.

As described above, from about 1 to 10 moles of derivatized drug areattached to each mole of antibody. In order to obtain this conjugationratio, it is usually necessary to use an excess quantity of a formulaIII derivatized drug. The reactivity of antibodies and active esters issomewhat variable but, in general, from about 5 to about 15 moles ofderivatized drug per mole of antibody are used in the process.

As a precautionary note, when the drug moiety of the derivatized drughas multiple reactive sites, it usually is necessary to block thesesites with protecting groups. Certain aspects of the use of protectinggroup has been discussed supra, and others are well known to theordinarily skilled organic chemist.

Products of this reaction are novel and are represented in formula Ibelow ##STR10## wherein R¹, R², m, and Ab are as defined above.

Finally, the immunoconjugate (drug-linker-antibody) is purified andisolated, usually by chromatographic methods. It may be possible toelute a conjugate from the chromatography medium in a concentrationwhich is appropriate for administration to patients. Customarily,however, the immunoconjugate will be purified by chromatography andeluted with any convenient solvent, in the highest concentration whichits solubility permits. A solvent solution containing 40% acetonitrileand 60% physiological phosphate buffered saline is preferred.

Compositions and Methods of Use

The immunoconjugates of the present invention are useful in thetreatment methods of the present invention, particularly whenparenterally administered in pharmaceutical compositions which are alsoan aspect of the present invention.

Such compositions, comprising an immunoconjugate of formula I and aparenterally-administrable medium, are formulated by methods commonlyused in pharmaceutical chemistry. For example, the presentimmunoconjugates are acceptably soluble in physiologically acceptablefluids (carriers) such as physiological saline solutions, serum proteinssuch as human serum albumin, buffer substances such as phosphates,water, and electrolytes, and the like.

Products for parenteral administration are often formulated anddistributed in a solid form preferably lyophilized, for reconstitutionimmediately before use. Such formulations are useful compositions of thepresent invention. Preparation of lyophilized compositions is well knownin the art. Generally, such compositions comprise mixtures of inorganicsalts which confer isotonicity, and dispensing agents, such as lactose,which allow the dried preparations to quickly dissolve uponreconstitution. Such formulations are reconstituted for use with highlypurified water.

The most effective concentration of the immunoconjugates of the presentinvention in a composition of the present invention is dictated by thedrug used in the conjugate, the physical properties of the drug andconjugate, and the final form of the composition. One skilled in the artof preparing such compositions will readily recognize the variables tobe considered and the optimal ratio of composition components.

Similarly, the most effective dosage regimen for the immunoconjugatecomposition of the present invention depends upon the severity andcourse of the disease/infection, the patient's health and response totreatment, and the judgment of the treating physician. Accordingly, thedosages of the immunoconjugates and any accompanying compounds should betitrated to the individual treatment. Otherwise, guidance to thespecific potencies of drugs and their appropriate dosage ranges is to beobtained from the standard medical literature.

The present invention also provides methods for treating susceptiblemammalian cells or tissues comprising administering an effective amountof an immunoconjugate of formula I above to a mammal in need of suchtreatment.

Furthermore, the present invention provides a method of inhibiting thegrowth of pathogens in a mammalian host comprising administering aneffective amount of an immunoconjugate of formula I above to a mammal inneed of such treatment.

Alternative embodiments of the methods of this invention include theadministration, either simultaneously or sequentially, of a number ofdifferent immunoconjugates bearing different drugs, or differentantibodies or antigen-recognizing fragments thereof, for use in methodsof combination chemotherapy.

For example, an embodiment of this invention may involve the use of anumber of desacetylvinblastine-immunoconjugates where the specificity ofthe antibody component of the conjugate varies, e.g., a number ofimmunoconjugates are used, each one having an antibody that bindsspecifically to a different antigen or to different sites or epitopes onthe same antigen present on the cell, tissue or pathogen of interest.

This embodiment may be especially useful in the treatment of certaintumors where the amounts of the various antigens on the surface of atumor is unknown or the tumor cell population is her erogenous inantigen expression and one desires to insure that a sufficient amount ofdrug is targeted to all of the cells at the tumor site. The use of anumber of immunoconjugates bearing different antigenic or epitopespecificities for the tumor increases the likelihood of obtainingsufficient drug at the tumor site. Additionally, this embodiment isimportant for achieving a high degree of specificity for the tumorbecause it is known in the art that the likelihood that normal tissuewill possess all of the same tumor-associated antigens is small [see,for example, Hellstrom, I., et al., J. Immunol., 127, 1: 157-160(1989)].

Alternatively, a number of different immunoconjugates can be used whereonly the drug component of the conjugate varies. For example, aparticular antibody can be linked to doxorubicin to form oneimmunoconjugate and can be linked to lometrexol to form a secondimmunoconjugate. Both conjugates can then be administered to a host tobe treated and will localize, because of the antibody specificity, atthe site of the selected target cell, tissue or pathogen to be treated.This embodiment may be important when administering an immunoconjugateto a cell, tissue or host bearing a pathogen to be treated where thetarget is known or suspected to be resistant to a particular drug orclass of drugs.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood, however, that these examples are only for illustrativepurposes and are not to be construed as limiting the scope of thisinvention in any manner.

EXAMPLE 1 ##STR11##

To a round bottom flask equipped with a stirrer, drying tube andtheromemeter, containing 50 mL of dichloromethane and 10 mL of drydimethyformamide (DMF), 3.8 grams of β-alanine benzyl ester HCl wasadded. When solution had been obtained, 1.9 gm of N-methylmorpholine wasadded and the mixture was cooled to less than 4° C. Next, 1.9 grams ofdiketene was dissolved in 25 mL of dichloromethane and the resultingsolution was added dropwise to the flask at a rate which allowed thetemperature to be maintained at less than 5° C. Following the additionof diketene, the reaction was stirred for 15 minutes, warmed to roomtemperature and again stirred for 1.5 hours. The reaction mixture wasconcentrated in vacuo over night and gave an orange viscous residuewhich was dissolved in ethyl acetate brine. The ethyl acetate layer wasseparated and sequentially washed with 10% citric acid solution, brine,saturated sodium carbonate, and brine. The resulting ethyl acetatesolution was dried over magnesium sulfate and concentrated in vacuo to apale orange liquid which was solidified upon standing at roomtemperature to give 4.18 grams (91%) of orange solid. NMR was used toconfirm the identity of the title compound.

EXAMPLE 2 ##STR12##

To a round bottom flask equipped with a stirrer, drying tube andthermometer, containing 4.7 mL of acetic anhydride and 3.3 mL oftriethylorthoformate, 2.0 grams of the product from Example 1 was addedand the solution was heated to reflux for 2 hours. Excess aceticanhydride and triethylorthoformate were removed in vacuo and a dark redviscous liquid remained. The red viscous liquid was dissolved in aminimum volume of ethyl acetate, and hexane was added to cloudiness.This mixture was poured into a 150 mL fritted glass Buchner funnel whichwas 75% full with silica gel 60. The gel was eluted with 500 mL of 20%ethyl acetate in hexane, 500 mL of 30% ethyl acetate in hexane, and 500mL of 40% ethyl acetate in hexane, and each fraction was collected. Thelast two fractions were combined and concentrated in vacuo to give 1.23grams (51%) of pale yellow syrup. The identify of the title compound wasconfirmed by NMR.

EXAMPLE 3 ##STR13##

Into a 50 mL round bottom flask equipped with a magnetic stirrer, wassuspended 5% palladium-on-carbon catalyst (Pd/C) in 10 mL of absoluteethanol. To this was added 1.2 grams of the reaction product fromExample 2 in 10 mL of absolute ethanol, followed by the addition of 0.7mL of 1,4-cyclohexadiene. After stirring the mixture for 30 minutes atroom temperature, the mixture was heated to 65° C. for 30 minutes. Afterthe Pd/C was removed by filtration, the residue was eluted with hotethyl acetate to give 0.28 grams (32%) of off-white crystals byfiltration. NMR was used to confirm the identity of the title compound.

EXAMPLE 4 ##STR14##

To a 10 mL round bottom flask equipped with a magnetic stirrer andcontaining 0.8 mL of dry DMF, 278.0 mg ofdesacetylvinblastine-hydrazide•H₂ SO₄ (DAVLB-hydrazide•H₂ SO₄) was addedand dissolved. To this solution was added 73.5 mg of the reactionproduct from Example 3 and also dissolved. The reaction was monitored byHPLC (Waters Radial pak C18, flow rate of 5 mL/minute, using 65%MeOH:35% 0.1M pH=7.0 KH₂ PO₄). HPLC showed that the title conjugate wasformed within 20 minutes.

EXAMPLE 5 ##STR15##

The reaction flask from Example 4 was fitted with a septum and dryingtube and cooled to less than -20° C. To the reaction flask whichcontained the solution from Example 4 was added 300 μL of a solution of91 mg of N-hydroxy-succinimide in 369.0 μL of dry DMF. The solution wasstirred for 10 minutes at -20° C. and then allowed to gradually warm toroom temperature. The solution was continuously stirred overnight. Thefollowing day, the DMF was removed in vacuo with heat, (less than 65°C.), and the resulting viscous orange liquid was sealed and stored atless than -4° C. for at least two days. Following removal from storage,the excess N-hydroxy-succinimide crystals were selectively removed bydissolving the viscous orange liquid in dichloromethane and filtering.The product remaining in solution was precipitated by the addition ofether, redissolved in dichloromethane and reprecipitated with etherthree times. Following centrifugation, the residue was redissolved indichloromethane, transferred to a round bottom flask and concentrated invacuo to give 370 mg of off-white solid. A small portion of the solidwas dissolved in DMF, treated with isopropylamine and analyzed by HPLCwhich showed clean conversion to the title product.

EXAMPLE 6 ##STR16##

Antibody VX007B is produced by a hybridoma which is a subclone derivedfrom the hybridoma producing the antibody KS1/4, which is discussedabove in the antibody section of this document. A 500.0 μL portion of asolution containing 9.9 mg (19.8 mg/mL) of that antibody in 0.34M sodiumborate was added to a 3.0 mL vial equipped with a magnetic stirrer. Tothis solution was added 40.5 μL of a mixture of 2.4 mg of the productfrom Example 5 in 177 μL of dry DMF to deliver 0.55 mg of the productfrom Example 5. This solution was stirred at room temperature for 20minutes and centrifuged for 5 minutes. A 1.0 mL portion of thesupernatant was applied to a phenylsuperose column [FPLC, HR 5/5(Pharmacia, Inc., Piscataway, N.J.)] equilibrated with 0.1Mphysiological phosphate buffered saline (ppbs), and eluted via a stepgradient with a solution containing 40% acetonitrile and 60% 0.1M ppbs.Three pooled fractions were collected. A 500 μL portion of each of the 3pools was purified on superose 12 (elution with 10% acetonitrile inppbs) and analyzed by ultraviolet (UV) spectrophotometry. UV analysisindicated a total recovery of 4.4 mg of conjugate (45%). The conjugationratio for the three pools was 3.3, 4.5, and 6.3 moles of drug (m) permole of antibody, respectively.

EXAMPLE 6A ##STR17##

The process of Example 6 was followed, starting with 9.9 mg of antibodyand 0.69 mg of the product from Example 5. The total recoveredimmunoconjugate amounted to 3.0 mg (30%) and the conjugation ratio forthe three pools were 4.1, 4.5 and 7.2 moles of drug (m) per mole ofantibody, respectively.

EXAMPLE 7 ##STR18##

A 1.0 mL portion of a solution containing 18.9 mg of antibody VX007B in0.1M phosphate buffer was added to a 3.0 mL vial equipped with amagnetic stirrer. To this solution was slowly added a mixture of 1.59 mgof the product from Example 5 in 81.1 μL of dry DMF. This solution wasstirred for two hours at room temperature and centrifuged for 10minutes. The supernatant was injected into a phenyl superose column asdescribed in Example 6 which was equilibrated with 0.1M ppbs, and elutedvia a step gradient with a solution containing 40% acetonitrile in 0.1Mppbs. One pooled fraction was collected. The fraction was filteredthrough a MILLEX-GV® (Millipore, Bedford, Mass.) 0.22 μ filter andsubmitted for UV analysis which indicated a total recovery of 10.4 mg(55%) and a conjugation ratio of 4.0 moles of drug (m) per mole ofantibody.

EXAMPLE 7A ##STR19##

The process of Example 7 was followed, except the phenylsuperose columnwas equilibrated with 0.1M acetate buffer, and eluted with a solutioncontaining 40% acetonitrile in 0.1M acetate buffer. Of the single pooledfraction, 2 mL was injected into a SEPHADEX G-25M® column (Pharmacia)and the collected pool fractions were submitted for UV analysis. The UVanalysis indicated a total recovery of 7.9 mg (42%) and the ratio was4.4 moles of drug (m) per mole of antibody.

EXAMPLE 8 ##STR20##

To a 10 mL round bottom flask equipped with a magnetic stirrer andsupplied with a nitrogen atmosphere, and containing 4 mL of DMF, wassuspended 200.0 mg of doxorubicin. To this suspension was added 0.75 mLof saturated sodium bicarbonate solution which gave a dark redhomogeneous solution. Next, 95 mg of the produce from Example 3 wereadded and the solution was allowed to stand at room temperature. Thereaction was monitored by HPLC [Waters C18 Radial pak column, flow rateof 5 mL/minute, using 65% MeOH:35% NaOAc solution (3% w/v)] and, afterthree hours, the title compound was identified. After storing thereaction mixture overnight at 4° C., the reaction mixture was allowed towarm to room temperature, added to 50 mL of water and twice washed with150 mL of ethanol. The resulting solution was then acidified to a pH of3.0 with 0.2N HCl and quickly extracted into two portions each of 150 mLof ethanol. The ethanol extracts were pooled dried over magnesiumsulfate and concentrated in vacuo to give 242.8 (97%) of dark red solid.HPLC analysis of this material confirmed the identity of the titlederivatized drug.

EXAMPLE 9 ##STR21##

To a 10 mL round bottom flask equipped with a magnetic stirrer suppliedwith a dry nitrogen atmosphere, and containing 4 mL of DMF, was added194.4 mg of the Doxorubicin derivative from Example 8. To this solutionwere added 256.4 mg of1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide•HCl and, 15 minuteslater, 153.94 mg of N-hydroxysuccinimide. The reaction was monitored byHPLC (Waters C18 radial pak column as previously described). Three hoursafter the addition of the last reactant, the reaction was complete andthe title derivatized drug was identified. Although the reaction mixturewas stirred overnight at room temperature under a nitrogen atmosphere,it is best to work up the reaction mixture after three hours.

For work up, the entire reaction mixture was extracted into ethylacetate with sodium chloride solution and twice washed with the sodiumchloride solution and once with water. The resultant mixture was driedover magnesium sulfate, filtered and concentrated in vacuo to give about250 mg of red residue. The residue was dissolved in 20 mL ofdichloromethylene and 50 mL of ethanol was added. The precipitate ofproduct which immediately formed was filtered and dried to give 174.2 mg(79%) of red powder. HPLC analysis of this powder confirmed the identityof the title derivatized drug.

EXAMPLE 10 ##STR22##

A 741.0 μL portion of a solution containing 10.0 mg (13.5 mg/mL) ofantibody CC83 (as discussed supra) in 0.1M physiological phosphatebuffered saline (ppbs) was added to a 3 mL round bottom flask equippedwith a magnetic stirrer. To this solution were added 60.1 μL of amixture of 1.5 mg of the product from Example 9 in 164.0 μL of dry DMFto deliver 0.55 mg of the product from Example 9. The reaction mixturewas slowly stirred for 1.5 hours at room temperature, transferred to acentrifuge tube and centrifuged for 10 minutes. The supernatant wasinjected into a phenylsuperose column (FPLC; HR 5/5) equilibrated with asolution which contained 62.5% 0.1M of ppbs and 37.5% of a buffercontaining 40% acetonitrile and 60% 0.1M ppbs. Three pooled fractionswere collected. The second and third fractions were pooled and a 2.0 mLportion of this pool was injected into a SEPHADEX G-25® column (HR16/50) equilibrated and eluted with 0.1M ppbs. The column was eluted,via a step gradient, with the solution containing 40% acetonitrile and60% 0.1M ppbs. The pooled fractions gave about 8.0 mL of orange solutionwhich was filtered through a MILLEX-GV® 0.22 μ filter and analyzed byUV. The UV analysis indicated a total recovery of 7.8 mg of conjugate(78%), and the conjugation ratio was 3.9 moles of drug (m) per mole ofantibody.

EXAMPLE 10A ##STR23##

The process of Example 10 was followed except for the buffer in whichthe antibody solution was prepared and the length of time the reactionmixture was stirred prior to centrifugation. Rather than using 0.1Mppbs, 0.34M borate buffer was employed. Also, the reaction mixture wasstirred for 20 minutes rather than 1.5 hours. UV analysis indicated atotal recovery of 8.7 mg of conjugate (87%), and the conjugation ratiowas 5.2 moles of drug (m) per mole of antibody.

EXAMPLE 11 ##STR24##

Antibody D612 is produced by a 658.0 μL portion of a solution containing10 mg (15.2 mg/mL) of antibody D612 in 0.1M ppbs was added to a 3 mLround bottom flask. To this solution were added 53.3 μL of a mixture of2.10 mg of the product from Example 9 in 204.0 μL of dry DMF to deliver0.55 mg of the product from Example 9. The reaction mixture was slowlystirred for 20 minutes at room temperature, transferred to a centrifugetube and centrifuged for 10 minutes. The supernatant was injected into aphenylsuperose column (FPLC; HR 5/5) equilibrated with a solution whichcontained 62.5% of 0.1M ppbs and 37.5% of a buffer containing 40%acetonitrile and 60% of 0.1M ppbs. The column was eluted, via a stepgradient, with the solution containing 40% acetonitrile and 60% of 0.1Mppbs. Three pooled fractions were collected. The second and thirdfractions were pooled and a 2.0 mL portion of this pool was injectedinto a SEPHADEX G-25M ® column (HR 16/50) equilibrated with 0.1M ppbs.The pooled fractions gave about 8.0 mL of orange solution which wasfiltered through a MILTEX-GV® 0.22 μ filter and analyzed by UV. The UVanalysis indicated a total recovery of 7.6 mg of conjugate (76%), andthe conjugation ratio was 4.1 moles of drug (m) per mole of antibody.

EXAMPLE 11A ##STR25##

The process of Example 11 was followed except for the buffer in whichthe antibody solution was prepared. Rather than using 0.1M ppbs, 0.34Mborate buffer was employed. UV analysis indicated a total recovery of6.1 mg of conjugate (61%), and the conjugate ratio was 6.1 moles of drug(m) per mole of antibody.

Test I UCLA/P3 Tumors in Mice

The conjugate of Example 6 was tested in vivo against xenografts of theUCLA/P3 lung adenocarcinoma in female Charles River nude mice. The testwas begun by subcutaneously implanting each mouse with 10⁷ UCLA/P3 tumorcells. On each of days 2, 5 and 7 after implantation, each mouse wasinjected with the conjugate or with the unconjugated drug as acomparative base. The conjugate was the product of Example 6 and thedrug was desacetylvinblastine-hydrazide•H₂ SO₄. The doses of drug alone,or as a component of the conjugate ranged from 0.5 to 3.0 mg/kg. Thesize of the tumor induced by implantation was measured, if possible, 14,21 and 28 days after implantation and the percent inhibition wascalculated. Each treatment group consisted of 5 mice.

The following table reports the activity of the drug and the conjugateas % inhibition of the tumors.

    ______________________________________    Days After % Inhibition of the Tumor    Implantation               Drug      Drug Dosage                                    Conjugate    ______________________________________    14          92*      3.0 mg/kg   100**    21         89                   100    28         69                   100    14         80        1.0 mg/kg  100    21         71                   100    28         64                   100    14         79        0.5 mg/kg  100    21         50                   100    28         48                   100    ______________________________________     *Mouse mortality 1/5     **Mouse mortality 2/5

Test II UCLA/P3 Tumors in Mice

The procedure from Test I was used to establish UCLA/P3 lungadenocarcinoma in female Charles River nude mice. On each of days 2, 5and 7 after implantation, each mouse was injected with the conjugate(VX007B-F(ab')2-desacetylvinblastine adduct from Example 5),desacetylvinblastine-hydrazide•H₂ SO₄ alone, or the i.v. excipientalone. To dosage of drug, either alone or as a component of theconjugate, range from 0.25 to 1.0 mg/kg.

The following table reports the activity of the conjugate, drug andcontrol as mean tumor mass (mg) of existing tumors measured 24 daysfollowing implantation.

    ______________________________________    Drug Dosage,                Mean Tumor Mass, mg    mg/kg       Conjugate    Drug    Control    ______________________________________    2.0         0            50      520    1.0         20           140     510    0.50        50           505     505    0.25        305          620     505    ______________________________________

We claim:
 1. A method of inhibiting the growth of neoplasms in amammalian host comprising parenterally administering an effective amountan immunoconjugate of the formula ##STR26## wherein R¹ is C₁ -C₄alkyl;R² is a monovalent drug derivative having a reactively-availableamino, hydroxy or thiol function; m is an integer from 1 to 10; and Abis an antibody, or antigen-binding fragment thereof, which specificallybinds an antigen associated with a cell, tissue, or neoplasm to whichdelivery of the drug is desirable, to such a mammalian host in need ofsaid treatment.
 2. The method of claim 1 wherein R² of theimmunoconjugate is desacetylvinblastine.